Determination of ¹³C/¹²C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes

Abstract

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values.

13C/12C Ratios of endogenous urinary steroids investigated for doping control purposes

“…One sample with a T/EpiT of 76 was below the 3‰ threshold because of depleted endogenous values (see below), but a doping offence could be proven by analysing different target compounds. [21] Only four samples (0.3%), with a T/EpiT ranging from 4 to 6, exhibit values beyond the WADA IRMS threshold; the two samples with T/EpiT 1.7 and 1.8 respectively were both forwarded for IRMS determination because of A concentrations above the threshold of 10 000 ng/mL and were supposed to be due to the intake of testosterone prohormones.…”

“…The distribution of the values (OHA-A) of this subpopulation was compared to a reference population. [21] The distributions were compared visually by a QQ plot. In addition, the Kolmogorov-Smirnov test was performed, where the distribution of the test samples was compared to a Gaussian with mean and standard deviation of the reference distribution.…”

“…In order to ensure that these differences are due to false negative samples, the T/EpiT group was further tested against a reference population of n = 61 subjects. [21] Figure between the cumulative distributions rapidly increases towards higher quantiles. The corresponding one-sided KolmogorovSmirnov test is highly significant (D = 0.217, p < 0.001).…”

“…The remainder did not differ significantly in values for OHA-A from the independent reference population determined with the described IRMS method. [21] Further investigations on the topic of normal distribution of DHEA concentrations in urine showed that this distribution is positive skewed and therefore all samples determined with IRMS fit perfectly in this distribution and indeed should not be considered as suspicious. [23] Hence, after testing for different gender distribution between the DHEA and the T/EpiT samples (p = 0.8; no difference could be found) the DHEA samples were used as a reference population with the benefit that these samples and the T/EpiT samples were measured over the same period of time under the same conditions while the mentioned reference population only covers a short time period of four months.…”

Fast IRMS screening of pseudoendogenous steroids in doping analyses

“…The described confirmatory methods [12][13][14][15][16] are laborious, requiring a purification step by liquid chromatography in order to guarantee extracts of adequate purity suitable for IRMS analysis, precluding the application of the method on a large scale.…”